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Engineering production of functional scFv antibody in E. coli by co-expressing the molecule chaperone Skp.

Wang R, Xiang S, Feng Y, Srinivas S, Zhang Y, Lin M, Wang S - Front Cell Infect Microbiol (2013)

Bottom Line: Amongst the four purified proteins, the Skp co-expressed scFv showed the highest solubility, and the binding activity to antigen TLH was 3-4 fold higher than the other three purified scFv.In fact, this scFv is specific for TLH and does not cross-react with other TLH-associated proteins and could be used to detect TLH directly in real samples.These results suggest that the pACYC-Duet-skp co-expression vector might be a useful tool for the production of soluble and functional scFv antibody.

View Article: PubMed Central - PubMed

Affiliation: The Ministry of Education Key Laboratory of Biopesticide and Chemical Biology, College of Life Sciences, Fujian Agriculture and Forestry University Fuzhou, China.

ABSTRACT
Single-chain variable fragment (scFv) is a class of engineered antibodies generated by the fusion of the heavy (VH) and light chains (VL) of immunoglobulins through a short polypeptide linker. ScFv play a critical role in therapy and diagnosis of human diseases, and may in fact also be developed into a potential diagnostic and/or therapeutic agent. However, the fact that current scFv antibodies have poor stability, low solubility, and affinity, seriously limits their diagnostic and clinical implication. Here we have developed four different expression vectors, and evaluated their abilities to express a soluble scFv protein. The solubility and binding activity of the purified proteins were determined using both SDS-PAGE and ELISA. Amongst the four purified proteins, the Skp co-expressed scFv showed the highest solubility, and the binding activity to antigen TLH was 3-4 fold higher than the other three purified scFv. In fact, this scFv is specific for TLH and does not cross-react with other TLH-associated proteins and could be used to detect TLH directly in real samples. These results suggest that the pACYC-Duet-skp co-expression vector might be a useful tool for the production of soluble and functional scFv antibody.

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ELISA Detection of TLH in Bacteria. Eight different bacterial strains (three TLH positive V. parahaemolyticus strains, three TLH negative Vibrio strains and two other E. coli strains) were cultured in APW medium without shaking for 16 h, and the supernatant of culture was used to coat the 96 wells plate at 4°C for overnight. The ELISA assay was determined using an anti 6×His tag antibody. (PC, positive control; NC, negative control).
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Figure 6: ELISA Detection of TLH in Bacteria. Eight different bacterial strains (three TLH positive V. parahaemolyticus strains, three TLH negative Vibrio strains and two other E. coli strains) were cultured in APW medium without shaking for 16 h, and the supernatant of culture was used to coat the 96 wells plate at 4°C for overnight. The ELISA assay was determined using an anti 6×His tag antibody. (PC, positive control; NC, negative control).

Mentions: We were interested in determining whether the co-expressed scFv could serve as a good antibody reagent to directly detect TLH from bacterial strains. In the first detection, three TLH positive V. parahaemolyticus strains, three TLH negative Vibrio strains and two other E. coli strains were used to identify the practicality and feasibility of co-expressed scFv. As shown in Figure 6, three V. parahaemolyticus strains gave the positive signal, and other five non V. parahaemolyticus strains all gave the negative results. To further detect the accuracy of co-expressed scFv, total 22 different bacterial strains (eleven different types V. parahaemolyticus strains, seven Vibrio strains from other species containing no TLH, and four other bacterial strains) were tested. Table 2 showed that all the eleven V. parahaemolyticus strains were successfully detected by Skp co-expressed scFv. In contrast, seven Vibrio strains from other species and four other bacterial strains of non-Vibrio all gave negative detection results. These results showed that this Skp co-expressed scFv could specifically recognize TLH positive V. parahaemolyticus strains, and might be used as a potential reagent for V. parahaemolyticus diagnosis.


Engineering production of functional scFv antibody in E. coli by co-expressing the molecule chaperone Skp.

Wang R, Xiang S, Feng Y, Srinivas S, Zhang Y, Lin M, Wang S - Front Cell Infect Microbiol (2013)

ELISA Detection of TLH in Bacteria. Eight different bacterial strains (three TLH positive V. parahaemolyticus strains, three TLH negative Vibrio strains and two other E. coli strains) were cultured in APW medium without shaking for 16 h, and the supernatant of culture was used to coat the 96 wells plate at 4°C for overnight. The ELISA assay was determined using an anti 6×His tag antibody. (PC, positive control; NC, negative control).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3818579&req=5

Figure 6: ELISA Detection of TLH in Bacteria. Eight different bacterial strains (three TLH positive V. parahaemolyticus strains, three TLH negative Vibrio strains and two other E. coli strains) were cultured in APW medium without shaking for 16 h, and the supernatant of culture was used to coat the 96 wells plate at 4°C for overnight. The ELISA assay was determined using an anti 6×His tag antibody. (PC, positive control; NC, negative control).
Mentions: We were interested in determining whether the co-expressed scFv could serve as a good antibody reagent to directly detect TLH from bacterial strains. In the first detection, three TLH positive V. parahaemolyticus strains, three TLH negative Vibrio strains and two other E. coli strains were used to identify the practicality and feasibility of co-expressed scFv. As shown in Figure 6, three V. parahaemolyticus strains gave the positive signal, and other five non V. parahaemolyticus strains all gave the negative results. To further detect the accuracy of co-expressed scFv, total 22 different bacterial strains (eleven different types V. parahaemolyticus strains, seven Vibrio strains from other species containing no TLH, and four other bacterial strains) were tested. Table 2 showed that all the eleven V. parahaemolyticus strains were successfully detected by Skp co-expressed scFv. In contrast, seven Vibrio strains from other species and four other bacterial strains of non-Vibrio all gave negative detection results. These results showed that this Skp co-expressed scFv could specifically recognize TLH positive V. parahaemolyticus strains, and might be used as a potential reagent for V. parahaemolyticus diagnosis.

Bottom Line: Amongst the four purified proteins, the Skp co-expressed scFv showed the highest solubility, and the binding activity to antigen TLH was 3-4 fold higher than the other three purified scFv.In fact, this scFv is specific for TLH and does not cross-react with other TLH-associated proteins and could be used to detect TLH directly in real samples.These results suggest that the pACYC-Duet-skp co-expression vector might be a useful tool for the production of soluble and functional scFv antibody.

View Article: PubMed Central - PubMed

Affiliation: The Ministry of Education Key Laboratory of Biopesticide and Chemical Biology, College of Life Sciences, Fujian Agriculture and Forestry University Fuzhou, China.

ABSTRACT
Single-chain variable fragment (scFv) is a class of engineered antibodies generated by the fusion of the heavy (VH) and light chains (VL) of immunoglobulins through a short polypeptide linker. ScFv play a critical role in therapy and diagnosis of human diseases, and may in fact also be developed into a potential diagnostic and/or therapeutic agent. However, the fact that current scFv antibodies have poor stability, low solubility, and affinity, seriously limits their diagnostic and clinical implication. Here we have developed four different expression vectors, and evaluated their abilities to express a soluble scFv protein. The solubility and binding activity of the purified proteins were determined using both SDS-PAGE and ELISA. Amongst the four purified proteins, the Skp co-expressed scFv showed the highest solubility, and the binding activity to antigen TLH was 3-4 fold higher than the other three purified scFv. In fact, this scFv is specific for TLH and does not cross-react with other TLH-associated proteins and could be used to detect TLH directly in real samples. These results suggest that the pACYC-Duet-skp co-expression vector might be a useful tool for the production of soluble and functional scFv antibody.

Show MeSH